High-efficiency CRISPR genome-editing tools for multiplex editing

Ready-to-transfect GeneArt CRISPR Nuclease mRNA circumvents time-consuming cloning steps needed when using CRISPR vector systems. Simply co-transfect Cas9 mRNA with in vitro transcribed guide RNA (IVT gRNA) or a synthetic gRNA expression cassette. Following transfection, the Cas9 protein is directed by gRNA to target specific genomic locus. This system allows multiplex genome editing, where multiple target gene sequences can be edited simultaneously in a single transfection reaction by adding multiple gRNAs. The system is versatile and simple to use, and changing target specificity only requires a change in the gRNA design.

How to obtain a gRNA sequence

Genome editing with CRISPR technology requires a noncoding guide RNA (gRNA) in order to cleave genomic DNA at a target sequence of interest.

Use our GeneArt CRISPR Search and Design Tool to search our database of >600,000 gRNA sequences specific to every gene in the human and mouse genomes. GeneArt predesigned gRNAs are optimized for gene knockout. Search results include recommendations based on minimizing potential off-target effects, potential binding sites, and exon maps with gRNA locations. Use this tool to analyze any sequence of interest and design unique CRISPR sequences.

Choose one of three options for making your gRNA:

1. The GeneArt Precision gRNA Synthesis Kit for transfection-ready gRNA in as little as four hours including template assembly.

3. Save time and effort and have our GeneArt custom services team design, synthesize, and purify in vitro transcribed (IVT) gRNA sequences for you. To obtain a services quotation, or to order, please contact our Custom Services department at 1-800-955-6288 x45682 or custom.services@thermofisher.com.

Efficient multiplexing system enables reduced hands-on time with a simple workflow

Simultaneously transfect up to 4 targets in a single well, and assess the cleavage efficiency of multiple genes simultaneously. The Cas9 mRNA can be used in multiplexing approaches with more than one GeneArt CRISPR Strings DNA fragment. Use this approach to determine which gRNA sequence works best for a particular target, or edit multiple genomic loci with one transfection.

Other potential applications of Cas9 mRNA include generation of transgenic model systems

While we haven’t tested the use of GeneArt CRISPR nuclease mRNA in microinjections or other in vivo–mediated delivery methods for transgenic model system generation, many citations show its use in in vivo applications in a wide variety of organisms, including mouse, zebrafish, and Drosophila. For microinjection experiments and mouse model generation we recommend testing at least 3 gRNAs and validating the gRNAs in cell line of choice. Using GeneArt CRISPR Strings™ DNA allows you to simultaneously screen multiple gRNA in a single transfection. Following validation you can clone your validated gRNA into an expression plasmid and make IVT gRNA template from sequence verified plasmids. See user manual for detailed instructions on generating synthetic PCR templates from gRNA expression plasmids.

Figure 6. Highly efficient multiplexed genome editing using Cas9 mRNA and in vitro transcribed gRNA. Shown here are the editing efficiencies from either cells transfected with an all-in-one plasmid or cells treated with just one target or simultaneously targeted with either double or triple targets. We also observe RelA-specific cleavage in all the samples co-transfected with RelA in either single or multiplex format (results not included).